Oligonucleotides labeled with stable isotopes and a method for detecting the same

ABSTRACT

Antisense oligonucleotide sequences which enable the measurement of the distribution and structure of antisense oligonucleotide drugs in the body, with lapse of time, and a method of detecting these sequences are provided. The antisense chains have a natural or non-natural nucleotide or peptide nucleic acid as a structural unit in which carbon atoms and nitrogen atoms are substituted by  13 C and  15 N, respectively, and the antisense chains can be detected by nuclear magnetic resonance spectroscopy (NMR) such as  15 N— 1 H or  13 C— 1 H hereto nuclear multiple quantum coherence spectroscopy.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of Japanese applicationserial no. 11 -094323, filed Mar. 31, 1999.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates to methods for detecting antisenseoligonucleotides and peptide nucleic acids which are labeled with stableisotopes, and foreign antisense chains.

2. Background of the Invention

In recent years, antisense drugs using antisense technology have drawnattention as therapeutic agents for diseases such as cancers, geneticdiseases and AIDS. An antisense drug refers to an oligonucleotide or thelike that has a sequence complementary (antisense) to a part of asequence of a specific gene which causes a certain disease. Whenintroduced into the body (target cells), the antisense drug forms aspecific double strand chain with mRNA, which is a transcription productof the causative gene, or a precursor thereof, to inhibit translation orprocessing of the precursor mRNA. Thus, this inhibition can control theonset of the disease. The efficacy of antisense drugs heavily depends onthe technique to deliver the oligonucleotides to the target site. Thisis because nucleases that decompose nucleic acids are generally presentin the body and digest the introduced oligonucleotides before they reachthe target site, which disables the formation of the complementarydouble strand chains at the target site, thus no effect can be obtained.For example, a nucleic acid incorporated into a cell by endocytosis isincorporated into a lysosome in the cell, and then decomposed by anuclease in the lysosome. Thus, the introduced oligonucleotide cannotform a double strand chain with the target transcription product in thenucleus or cytoplasm, resulting in no effect. An example of a usefuldelivery technique to solve this problem is the use of intracellularrouting agents, typically represented by a liposome preparation, whichhas provided a certain level of success.

Another attempt to improve the effect of antisense drugs involvesincreasing the stability of the antisense chains by modifying thenucleotides, the structural units of oligonucleotides, to non-naturalmodified nucleotides which are less susceptible to decomposition bynucleases.

In developing therapeutic drugs, pharmacokinetic tests for absorption,metabolism and excretion of the drugs are carried out. Inpharmacokinetic tests, substances to be tested (drugs) are labeled withradioactive isotopes or the like and administered to experimentalanimals, and the concentration and radioactivity of the drugs arequantitatively measured with the lapse of time. Antisense drugs are noexception and antisense oligonucleotides labeled with radioactiveisotopes or the like are subjected to pharmacokinetic tests.

In the use of antisense oligonucleotide drugs, the sequence and lengthof the nucleotides have to be conserved in body cells to fully implementtheir function. However, conventional pharmacokinetic tests usingradioisotopes or the like provide information on the distribution in thebody, absorption rate, and excretion rate of the oligonucleotides butnot on the conservation of their sequence and length. Accordingly, thestate of the conservation of oligonucleotides has to be confirmed byextracting a nucleic acid fraction from blood, organs or the like takenfrom experimental animals to analyze oligonucleotides in the fraction byhigh performance liquid chromatography, Southern blotting, Northernblotting, capillary electrophoresis, or the like. Furthermore, it wasvirtually impossible to see the change in the length of the administeredantisense nucleotides with the lapse of time (the progress ofdecomposition). Further, there is no means to confirm whether the targetmRNA or precursor mRNA and the oligonucleotide introduced into cellsform complementary double strand chains. Moreover, the efficacy of theintroduction of the oligonucleotide can only be judged cytologicallybased on physiological or morphological changes of the cells to whichthe oligonucleotide is introduced. Furthermore, in conventionalpharmacokinetic tests, the use of radioisotopes is inevitable, whichrequires a special facility according to specified regulations andwell-trained technicians.

SUMMARY OF THE INVENTION

The present invention was made in consideration of the abovementionedproblem and an objective of the present invention is to provideantisense oligonucleotide sequences and antisense peptide nucleic acidsequences labeled with stable isotopes, which enables the measurement ofthe distribution, state of conservation and structure of antisenseoligonucleotide drugs in the body, with the lapse of time, and a methodof detecting these sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows 500 MHz ¹H-NMR spectra of non-exchangeable protons forsamples 1 to 4.

FIG. 2 shows 500 MHz ¹H-NMR spectra of exchangeable protons for samples1 to 4.

FIG. 3 shows 500 MHz ¹H-NMR spectra of non-exchangeable protons forsample 1.

FIG. 4 shows 500 MHz ¹H-NMR spectra of exchangeable protons for sample1.

FIG. 5 shows 500 MHz ¹³C—¹H HMQC spectra of non-exchangeable protons forsample 1.

FIG. 6 shows 500 MHz ¹H-NMR spectra of non-exchangeable protons forsample 2.

FIG. 7 shows 500 MHz ¹H-NMR spectra of exchangeable protons for sample2.

FIG. 8 shows 500 MHz ¹H-NMR spectra of non-exchangeable protons forsample 3.

FIG. 9 shows 500 MHz ¹H-NMR spectra of exchangeable protons for sample3.

FIG. 10 shows 500 MHz ¹³C—¹H HMQC spectra of non-exchangeable protonsfor sample 3.

FIG. 11 shows 500 MHz ¹⁵N—¹H HMQC spectra of non-exchangeable protonsfor sample 3.

FIG. 12 shows 500 MHz ¹H-NMR spectra of non-exchangeable protons forsample 4.

FIG. 13 shows 500 MHz ¹H-NMR spectra of exchangeable protons for sample4.

FIG. 14 shows 500 MHz ¹³C—¹H HMQ spectra of non-exchangeable protons forsample 4.

FIG. 15 shows 500 MHz ¹⁵N—¹H HMQ spectra of non-exchangeable protons forsample 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to solve the abovementioned problem and attain theabovementioned objective, the present invention is constituted asfollows. Namely, an antisense oligonucleotide sequence and antisensepeptide nucleic acid sequence of the present invention are single strandRNAs or single strand or double strand DNAs comprising at least onenucleotide or peptide nucleic acid as a structural unit, in which atleast one structural C atom is substituted with ¹³C, and at least onestructural N atom is substituted by ¹⁵N, having 10 to 100 basescomplementary to the desired target sequence to be hybridized. In theseantisense oligonucleotide sequence and antisense peptide nucleic acidsequence, said oligonucleotide is

1) a natural oligonucleotide wherein 3′-OH and 5′-OH in ribose ordeoxyribose are cross-linked by phosphodiester bonds,

2) a phosphorothioate oligonucleotide wherein one or twonon-cross-linked oxygen atoms in the phosphodiester bonds in saidnatural oligonucleotide are substituted by sulfur atoms, or

3) a methylphosphonate oligonucleotide wherein oxygen atoms in thehydroxyl groups in the phosphodiester bonds in said naturaloligonucleotide are substituted by methyl groups, and

said peptide nucleic acid has bases, i.e., purine or pyrimidine, andsaid bases are linked together by peptide bonds to form a2-aminoethylglycine backbone.

In an antisense oligonucleotide sequence and an antisense peptidenucleic acid sequence of the present invention, it is preferable thatall carbon atoms are substituted by ¹³C, and all nitrogen atoms aresubstituted by ¹⁵N in all structural units, i.e., nucleotides andpeptide nucleic acids.

A composition of the present invention comprises the abovementionedantisense oligonucleotide sequence or antisense peptide nucleic acidsequence, and a pharmaceutically acceptable carrier.

A method of detecting an antisense oligonucleotide sequence containingstable isotopes or decomposition products thereof, or an antisensepeptide nucleic acid sequence containing stable isotopes ordecomposition products thereof, comprises

a step of sampling at least one material to be measured, i.e., blood,tissues, organs, body fluids, cells or excretions from a subject animalto which an antisense oligonucleotide sequence or antisense peptidenucleic acid sequence having a sequence complementary to a desiredtarget sequence to form a hybrid is administered, or a step of samplingsubject culture cells to which an antisense oligonucleotide sequence orantisense peptide nucleic acid sequence having a sequence complementaryto a desired target sequence to form a hybrid is administered, and

a step of measuring the antisense oligonucleotide or antisense peptidenucleic acid derived from said sampled material to be measured, bynuclear magnetic resonance spectrometry,

said antisense oligonucleotide sequence or antisense peptide nucleicacid sequence being the abovementioned antisense oligonucleotidesequence or antisense peptide nucleic acid sequence of the presentinvention.

A method of detecting an oligonucleotide sequence containing stableisotopes and decomposition products thereof, or an antisense peptidenucleic acid sequence containing stable isotopes and decompositionproducts thereof comprises a step of subjecting a subject animaladministered with the abovementioned antisense oligonucleotide sequenceor antisense peptide nucleic acid sequence of the present invention tomagnetic resonance imaging.

An antisense oligonucleotide sequence and antisense peptide nucleic acidsequence of the present invention are explained as follows.

The nucleotide sequence and peptide nucleic acid (hereinafteroccasionally referred to as an antisense chain in this invention), thestructural unit of an antisense oligonucleotide sequence and antisensepeptide nucleic acid sequence respectively of the present invention, isa natural nucleotide or non-natural nucleotide, or a peptide nucleicacid. Examples of a natural nucleotide include purine nucleotides, suchas adenosine and guanosine nucleotide, and pyrimidine nucleotides, suchas thymidine, uridine and cytidine nucleotide. Examples of a non-naturalnucleotide include phosphorothioate nucleotides, in which one or twooxygen atoms of the phosphoryl group are substituted by sulfur atoms,and methylphosphonate nucleotides, in which an oxygen atom of a hydroxylgroup in the phosphoryl group is substituted by a methyl group, andpeptide nucleic acids, artificial nucleotides, in which a base isintroduced into a 2-aminoethyl glycine framework.

Furthermore, in these structural units, at least one carbon atom, astructural atom, is substituted by ¹³C, and at least one nitrogen atom,a structural atom, is substituted by ¹⁵N. By substituting carbon atomsin an antisense chain, the presence of the antisense chain in a materialto be measured can be confirmed by nuclear magnetic resonancespectrometry such as ¹³C—¹H heteronuclide multiple quantum coherencespectrometry (hereinafter referred to as HMQC method) and ¹³C—¹Hheteronuclide single quantum coherence spectrometry (hereinafterreferred to as HSQC method). Further, by substituting nitrogen atoms inan antisense chain, whether the antisense chain has formed base pairscan be confirmed by nuclear magnetic resonance spectrometry such as¹⁵N—¹H HMQC method and ¹⁵N—¹H HSQC method).

A target of an antisense chain of the present invention is a knownsequence and can be appropriately selected depending on the purpose. Inconsideration of the best balance between the cost for the synthesis ofthe antisense chain, and the capability for stable hybridization withthe target sequence, the length of the antisense chain is preferably 10to 100 bases, more preferably 15 to 50 bases. If the length of theantisense chain is less than 10 bases, stable hybridization with thetarget sequence in vitro is rather difficult. On the other hand, if thelength of the antisense chain is more than 100 bases, the synthesis ofthe antisense chain containing stable isotopes costs a great deal, whichis not preferable. An antisense chain of the present invention issynthesized using a sequence, in which carbon atoms and nitrogen atomsof the structural unit, i.e., a nucleotide or peptide nucleic acid, aresubstituted by concentrated ¹³C and ¹⁵N, according to a method known toone skilled in the art. The structural unit used in the synthesis isavailable, for example, from Nippon Sanso Corporation, and a method forthe synthesis is described in detail in Japanese Patent Laid-open No.1994-319581 and Japanese Patent Laid-open No. 1995-115987. Accordingly,an antisense chain of the present invention synthesized by this methodcontains ¹³C and ¹⁵N in a higher ratio than the corresponding naturalchain. It is preferable that more than 90% of the carbon atoms andnitrogen atoms in the molecule are substituted by ¹³C and ¹⁵N.

In the antisense oligonucleotide sequence or antisense peptide nucleicacid sequence, it is preferable that structural atoms, i.e., carbonatoms and nitrogen atoms, in every structural unit (nucleotide orpeptide nucleic acid) are substituted by ¹³C or ¹⁵N. By substituting allcarbon atoms by ¹³C, signals arising from a ¹³C-labeled antisense chaincan be selectively detected without detecting many other substances inwhich structural carbon atoms are ¹²C. On the other hand, bysubstituting all nitrogen atoms by ¹⁵N, signals arising from ¹⁵N-labeledantisense chains can be selectively detected without detecting manyother substances in which structural nitrogen atoms ate ¹⁴N.Furthermore, when labeled by ¹⁵N, an imino group proton for each basepair can be detected and distinguished, which can provide information onhydrogen bonding between each base (secondary structure).

A method for detecting an antisense chain of the present invention willbe explained as follows.

First, at least one material to be measured, i.e., blood, tissues,organs, body fluids, cells or excretions, is sampled from a subjectanimal to which an isotope-labeled antisense oligonucleotide sequence orantisense peptide nucleic acid sequence having a sequence complementaryto the desired target sequence to form hybrid, or from subject culturecells to which the similar sequence has been administered. A method ofadministering the antisense chain to the subject animal or subjectculture cells is not particularly restricted, and various intracellularrouting agents, such as liposome, e.g., lipofectin, and various drugdelivery systems, such as a system using a histone subunit can be used.The antisense chain to be administered is not restricted to theisotope-labeled antisense oligonucleotide sequence and antisense peptidenucleic acid sequence, and compositions comprising these sequences and apharmaceutically acceptable carrier can be used. Examples of apharmaceutically acceptable carrier include liposomes such as lipofectinand nucleotide binding proteins such as histone protein.

The time of sampling of the material to be measured from the subjectanimal or subject culture cells are determined depending on the purpose.Namely, the sampling can be carried out immediately after theadministration of the antisense chain, or approximately several to 10days after the administration, taking into consideration the timerequired to release the antisense chain from liposome or the like intothe cells.

The material to be measured is then measured by nuclear magneticresonance spectrometry. In some cases, an appropriate pretreatment iscarried out. For example, if the material to be measured is a fluid,such as blood or a body fluid, it is preferable to carry out nuclearmagnetic resonance spectrometry after removing solids by centrifugation.If the sampled material to be measured is a solid, such as tissues orcells, it is necessary to homogenize the tissues and cells in a buffersolution, in which pH, salt concentration, and the like are adjusted, toextract a nucleic acid component, before the measurement by nuclearmagnetic resonance spectrometry. The buffer solution and conditions forthe homogenization can be appropriately determined by one skilled in theart depending on the material to be measured.

In the measurement by nuclear magnetic resonance spectrometry, it ispreferable that the concentration of the antisense chain to be measuredis 0.1 mM to 10 mM, and the concentration is appropriately adjustedbefore the measurement. About 5 to 10% of heavy water must be added tothe pretreated material to be measured for the lock signal.

Nuclear magnetic resonance spectrometry is preferably performed by theone-dimensional or two-dimensional measurement using either HMQC methodor HSQC method for ¹⁵N—¹H and ¹³C—¹H. In this case, it is morepreferable to carry out coherence selection by the pulse field gradient(PFG) method, or to remove solvent signals.

Measurement of ¹³C—¹H by HMQC method or HSQC method indicates whetherthe antisense chain is contained in the material being measured, and ifcontained, the concentration can be determined. Measurement of ¹⁵N—¹H byHMQC method or HSQC method indicates whether base pairs are formed inthe material being measured. Using these methods, a pre-measuredspectrum of the whole-length antisense chain and a spectrum of theantisense chain in the material sampled from the subject animal arecompared to analyze whether the antisense chain being measured has beenshortened by decomposition, or has been maintained over its wholelength. Furthermore, a pre-measured spectrum of a double strand chain ofthe whole-length antisense chain with a target sequence, which is formedin vitro, and a spectrum of the antisense chain in the material sampledfrom the subject animal are compared to determine whether the antisensechain being measured forms a double strand chain with the targetsequence. Further, combination with a quantitative analysis canadvantageously make the analysis more sensitive.

In the present invention, magnetic resonance imaging can be used insteadof the abovementioned nuclear magnetic resonance spectrometry. Anantisense chain can be administered to a subject animal in the samemanner as for nuclear magnetic resonance spectrometry. Afteradministration, the subject animal or subject culture cell conjugate issubjected to magnetic resonance imaging devise for the measurementwithout the isolation of the material to be measured from the subjectanimal, or any pretreatment. In particular, when an animal is used asthe subject, the state of absorption, secretion and excretion of theantisense chain in the body can be quantitatively observed and monitoredwith the lapse of time.

An antisense chain and a method for detecting the antisense chain can beused in various ways besides the abovementioned pharmacokinetic test.Examples of other uses include the diagnosis and treatment of diseases,such as cancers, genetic diseases, AIDS and influenza, and theevaluation of novel intracellular routing agents and drug deliveringsystems.

When applied to the diagnosis of diseases, the detection method of thepresent invention can be carried out in the same manner as forpharmacokinetic tests. Nuclear magnetic resonance spectrometry can beused to measure whether the antisense chain forms a double strand chainwith a target sequence in cells or blood in cases where the diagnosiswill be made using sample cells or blood that can be extracted from thebody. Nuclear magnetic imaging can be used to measure whether theantisense chain forms a double strand chain with a target sequence intissues or organs in cases where the diagnosis will be made usingtissues and organs that cannot be excised from the body,

For the treatment of the abovementioned diseases, an antisense chain ofthe present invention can be appropriately used to confirm whether thetreatment is effective or not, by nuclear magnetic resonancespectrometry or magnetic resonance imaging. Accordingly, the presentinvention can be used in treating diseases, and is useful in determiningthe amount and interval of administration of the antisense drug topatients.

In developing a novel intracellular routing agent or drug deliverysystem, an evaluation of its effectiveness is necessary. Theeffectiveness of the drug introduction can be evaluated by the detectionmethod of the present invention using an antisense chain of the presentinvention.

EXAMPLES

An experiment was carried out to confirm whether a RNA labeled withstable isotopes was detectable by NMR when the RNA was administered tomice.

Materials

Four female SPF/VAF mice (BALB/cAnNCr, 8 weeks old, No. 1 to No. 4)supplied by Charles River Japan, Inc. were used for the experiment.

A nucleotide labeled with ¹³C and ¹⁵N was synthesized by the methoddescribed in Japanese Patent Laid-open No. 1994-319581 and JapanesePatent Laid-open No. 1995-115987. An outline of this synthesizing methodwill be described as follows.

First, yeast cells of Candida utilis IFO-0369 were cultured in aninorganic medium using ¹³C-labeled acetic acid (¹³CH₃ ¹³COOH) as acarbon source and ¹⁵N-labeled ammonium chloride (¹⁵NH₄Cl) as a nitrogensource, and then harvested. Cell walls were decomposed by a cell walllytic enzyme, zymolyase (Kirin Breweries, Ltd.), after which thesupernatant obtained by centrifugation was further centrifuged (100,000g×3 hours) to obtain a ribosome fraction as a precipitate. Then, thisribosome fraction was treated with an equivalent amount of an aqueoussaturated phenol solution to remove proteins, and then ethanol was addedto precipitate a ¹³C— and ¹⁵N-labeled RNA.

The precipitate was washed with ethanol and dried under vacuum to obtain¹³C— and ¹⁵N-labeled RNA of greater than 90% purity. This RNA wasdecomposed into ribonucleotide 5′-phosphates using nuclease P1 (YamasaShoyu Co.), and then the ribonucleotide 5′-phosphates were fractionatedwith an anion exchange column, AG1X8 formic acid-type (BioRad), toobtain AMP, CMP, UMP and GMP.

Next, two phosphate groups were enzymatically added to each of theabovementioned AMP, CMP, UMP and GMP using phosphoenol pyruvic acid as aphosphate donor to obtain ribonucleotide 5′-triphosphates labeled with¹³C and ¹⁵N, according to the method of Whitesides et al. (J. Org. Chem.55, 1834-1841, 1990).

The oligonucleotide comprising 43 bases shown in SEQ ID No. 1 wassynthesized using the abovementioned ribonucleotide 5′-triphosphatelabeled with ¹³C and ¹⁵N, as a substrate. Synthesis was carried outusing a synthesized DNA (a product of Hokkaido System Science; purifiedby HPLC) as a template, and T7RNA polymerase (Air Brown). Aftersynthesis, the reaction solution was subjected to polyacrylamide gelelectrophoresis for purification, and the resulting target band was cutwith the gel from which RNA was extracted. The extracted RNA was twiceprecipitated with ethanol to remove salts, and the RNA labeled with 13Cand ¹⁵N was obtained.

A mixture of histone subunits H2A, H2B, H3 and H4 (Boehringer MannheimGmbH) was used as a drug delivery system.

Experimental Method

The abovementioned oligonucleotide (6 mg) was dissolved in 120□1 of aphysiological saline solution to prepare four 30□1 samples. Two sampleswere administered without a drug delivery system. The abovementionedhistone subunit mixture was added to the remaining two samples to form acomplex.

Sample 1: ¹³C—¹⁵N-labeled RNA (inoculated into and sampled from No. 1mouse)

Sample 2: ¹²C—¹⁵N-labeled RNA (inoculated into and sampled from No. 2mouse)

Sample 3: the complex of ¹³C—¹⁵N-labeled RNA and the drug deliverysystem (inoculated into and sampled from No. 3 mouse)

Sample 4: the complex of ¹²C—¹⁵N-labeled RNA and the drug deliverysystem (inoculated into and sampled from No. 4 mouse)

Each sample solution (100□1), which was adjusted to 62.5 mg/kg, wasinoculated into the tail of the abovementioned mice. Three hours afterthe inoculation, the mice were anesthetized with ether and about 1 ml ofwhole blood was sampled from the heart. This whole blood was allowed tostand at room temperature for 30 minutes, and then centrifuged at 1,500rpm at room temperature for 15 minutes. Supernatant serum obtained bythe centrifugation was subjected to NMR measurement as follows.

Heavy water (20□1) was added to the samples (400□1 each) obtained by theabovementioned centrifugation, and the measurement of NMR spectra wascarried out at 25C. A DRX-500 NMR spectrometer (BRUKER) was used for themeasurement. Pre-saturation was carried out for the measurement ofnon-exchangeable protons and jump-and-return pulses were used for themeasurement of exchangeable imino proton spectra.

Results of the Measurements

FIGS. 1 to 15 show results of the measurements. The stableisotope-labeled oligonucleotide of the present invention was barelydetected in all cases when no complex with the drug delivery system wasformed, while the entire length of the oligonucleotide was detected whena complex with the drug delivery system was formed. Details will beexplained referring to the drawings.

Evaluation

FIG. 1 shows the spectra of non-exchangeable protons for samples 1 to 4measured at 500 MHz-¹H. Marks 1 to 4 designate samples 1 to 4,respectively. Signals derived from nucleic acid bases were observedmainly in the range of 9 to 6.5 ppm. A close look of this range showsthat only extremely weak signals were detected for samples 1 and 2 whichwere administered without the drug delivery system. On the other hand,distinctive signals were detected in this range for samples 3 and 4which were administered with the drug delivery system. Therefore, it isobvious that the antisense chain existed in the blood for up to 3 hourswhen the drug delivery system was used.

FIG. 2 shows the spectra of exchangeable protons (imino protons) forsamples 1 to 4 measured at 500 MHz-¹H. Marks 1 to 4 designate samples 1to 4, respectively. Signals derived from base pairs, which werestabilized by forming a secondary structure, were observed in the rangeshown in this figure. Absolutely no signal was detected for samples 1and 2, but a set of strong, sharp signals were observed for samples 3and 4. Therefore, these results suggest that the RNA present in theblood did not undergo partial decomposition when the drug deliverysystem was used. Such sharp signals would not have been observed and thespectra should have been more complex had the RNA undergone partialdecomposition.

FIGS. 3, 6, 8 and 12 show the 500 MHz ¹H-NMR spectra of non-exchangeableprotons for samples 1 to 4. Comparison of these spectra shows that RNAwas barely detected for samples 1 and 2, but clearly detected forsamples 3 and 4. Comparison of the top and the bottom of FIGS. 3 and 4shows that the spectrum in the range between 9 and 6.5 ppm was changedby decoupling, which indicates that the signals in this range werederived from the labeled RNA. Further, the signals observed at 8.4 ppmin FIG. 8 and FIG. 12 were not changed by decoupling, which suggeststhat the signals were derived from the drug delivery system.

FIGS. 4, 7, 9 and 13 show the 500 MHz ¹H-NMR spectra of exchangeableprotons for samples 1 to 4. Comparison of these spectra shows that theRNA was barely detected for samples 1 and 2, but clearly detected forsamples 3 and 4. Comparison of the top and the bottom of FIGS. 9 and 13shows that the entire spectrum in this range was changed by decoupling,which shows that the signals in this range were derived from the labeledRNA.

FIGS. 5, 10 and 14 show the ¹³C—¹H HMQC spectra of non-exchangeableprotons for samples 1, 3 and 4. The vertical axis is the ¹³C chemicalshift and the horizontal axis is the ¹H chemical shift. Only noise, andno spectrum derived from RNA, was observed for sample 1. On the otherhand, spectra for samples 3 and 4 showed typical RNA patterns.

FIGS. 11 and 15 show the ¹⁵N—¹H HMQC spectra of non-exchangeable protonsfor samples 3 and 4. The vertical axis is the ¹⁵N chemical shift and thehorizontal axis is the ¹H chemical shift. These figures show that themeasured RNA was chemically and structurally homogeneous.

Effectiveness of the Invention

Since the antisense chain of the present invention has a natural ornon-natural nucleotide or peptide nucleic acid as a structural unit andcarbon atoms or nitrogen atoms are substituted by stable isotopes, i.e.,¹³C or ¹⁵N, the antisense chain can be quantitatively measured, with thelapse of time, by nuclear magnet resonance spectrometry such as ¹⁵N—¹Hand ¹³C—¹H heteronuclide multiple quantum coherence spectrometry, ormagnetic resonance imaging according to the detection method of thepresent invention, and the formation of double strand chains can also bedetected. Furthermore, the distribution of the antisense chain in thebody, or the like, can be analyzed, with the lapse of time, withouthaving to sample a material from the subject animal.

1-10. (canceled)
 11. An antisense drug using an oligonucleotide sequenceor peptide nucleic acid sequence comprising a single strand RNA orsingle strand DNA containing at least one nucleotide or peptide nucleicacid as a structural unit, in which at least one structural C atom issubstituted by ¹³C, and at least one structural N atom is substituted by¹⁵N, having 10 to 100 bases, said oligonucleotide being i) aphosphodiester oligonucleotide wherein 3′-OH and 5′-OH in ribose ordeoxyribose are cross-linked by phosphodiester bonds, ii) aphosphorothioate oligonucleotide wherein one or two non cross-linkedoxygen atoms in the phosphodiester bonds in said phosphodiesteroligonucleotide are substituted by sulfur atoms, or iii) amethylphosphonate oligonucleotide wherein oxygen atoms in the hydroxylgroups in the phosphodiester bonds in said phosphodiesteroligonucleotide are substituted by methyl groups, and said peptidenucleic acid having bases, i.e., purine or pyrimidine, and said basesbeing linked together by peptide bonds to form a 2-aminoethylglycinebackbone, wherein more than 90 % of the carbon atoms are substituted by¹³C and more than 90 % of the nitrogen atoms are substituted by ¹⁵N inevery structural unit, i.e., nucleotide or peptide nucleic acid.
 12. Anantisense drug using an oligonucleotide sequence or peptide nucleic acidsequence according to claim 11, wherein all carbon atoms are substitutedby ¹³C and all nitrogen atoms are substituted by ¹⁵N in every structuralunit, i.e., nucleotide or peptide nucleic acid.
 13. A method ofdetecting an antisense drug using an oligonucleotide sequence containingstable isotopes or a peptide nucleic acid sequence containing stableisotopes, or decomposition products thereof, comprising a step ofsubjecting a subject animal, to which the antisense drug using theoligonucleotide sequence or peptide nucleic acid sequence according toclaim 11 is administered, to magnetic resonance imaging.
 14. A method ofdetecting an antisense drug using an oligonucleotide sequence containingstable isotopes or a peptide nucleic acid sequence containing stableisotopes, or decomposition products thereof, comprising a step ofsubjecting a subject animal, to which the antisense drug using theoligonucleotide sequence or peptide nucleic acid sequence according toclaim 12 is administered, to magnetic resonance imaging.